Stimulatory effect of bleomycin on the hyaluronic acid synthetase in cultured fibroblasts

Biochemical Pharmacology. Vol. 27, pp. 1551-1554. © Pergamon Press Ltd. 1978. Printed in Great Britain.

0006-2952/78/0601-1551/$02.(~)/0

STIMULATORY E F F E C T OF BLEOMYCIN ON T H E H Y A L U R O N I C ACID S Y N T H E T A S E IN C U L T U R E D FIBROBLASTS K A T S U H I R O O T S U K A , S E I - I T S U M U R O T A and Y o M O R I * Department of Biochemistry, Tokyo College of Pharmacy, Hachioji-shi, Tokyo 192-03 ( K. O. and Y. M. ), and Department of Pharmacology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173 (S. M.), Japan

(Received 28 July 1977; accepted 12 September 1977) Abstract--The mechanism of the stimulatory effect of bleomycin on the production of hexosaminecontaining substances by cultured fibroblasts was studied by measuring changes in the activity of hyaluronic acid (HA) synthetase. HA synthetase activity was increased by I /zg bleomycin/ml, and its stimulatory effect could be detected 4 days after the start of bleomycin treatment and was enhanced by lengthening the time of treatment, i.e. by 96 per cent for an 8-day treatment. In cell-free systems, however, bleomycin showed no stimulatory effect on this enzyme activity. The enhancement of HA synthetase activity by bleomycin was inhibited by the addition of cycloheximide or actinomycin D. The synthesis of HA was much less influenced by cycloheximide than was that of glycoprotein and sulfated glycosaminoglycan. From our results it can be concluded that HA synthetase activity, induced when the old medium of stationary cultures was renewed with fresh medium containing 5% fetal bovine serum, was significantly enhanced by the addition of bleomycin. However, the detailed mechanism of enhancement of HA synthetase activity has not yet been elucidated. It is well k n o w n that bleomycin, an antitumor antibiotic isolated f r o m a strain of Streptomyces verticillus [ i ], has a variety of actions such as singlestrand breaks of D N A , inhibition of D N A - d e p e n d e n t D N A p o l y m e r a s e , and arrest of cells just prior to mitosis [2-7]. In p r e v i o u s papers [8, 9], we r e p o r t e d that bleomycin signifcantly increased the synthesis of acidic g l y c o s a m i n o g l y c a n s ( A G A G ) , g l y c o p r o t e i n s and collagen in cultured fibroblasts. H o w e v e r , since these e x p e r i m e n t s w e r e p e r f o r m e d in the steady state, it was possible that the incorporation of labeled thymidine into D N A was inhibited in only a small p e r c e n t a g e of cells, w h e r e a s A G A G was synthesized in about 95 per c e n t of cells. Accordingly, we suggested that inhibition of D N A synthesis is i n d e p e n d e n t of the stimulatory effect of b l e o m y c i n on A G A G synthesis. In this report, the effect of b l e o m y c i n on H A synthetase was studied by using cultured fibroblasts k n o w n to produce primarily a large a m o u n t of H A [9]. EXPERIMENTAL

Culture conditions. Cloned fibroblasts (clone C5 f r o m rat carrageenin granuloma) w e r e used [ 10, 11 ]. The cells w e r e g r o w n in a Petri dish at 37 ° in a CO2 a t m o s p h e r e with H a m ' s F-12 m e d i u m (Nissui Seiyaku C o . , T o k y o ) s u p p l e m e n t e d with 10% fetal bovine serum (Microbiological Associates, B e t h e s d a , MD) and antibiotics (penicillin, 100 units/ml, and s t r e p t o m y c i n , 100/zg/ml). All experiments started just after the cells r e a c h e d their stationary phase, w h e n the c o n c e n t r a t i o n of serum * To whom requests for reprints should be addressed. 1551

was lowered to 5% so that a constant cell n u m b e r was maintained. A more detailed description is given in a previous paper [9]. Treatment of cells. A sterilized aqueous solution of b l e o m y c i n hydrochloride (Lot No. 1600AC01, a gift f r o m Nippon K a y a k u C o . , Tokyo) was stored as a stock solution (i mg/ml) at - 2 0 ° until use. Just before use, the stock solution was diluted with H a m ' s F-12 medium, containing 5% fetal bovine serum, to the appropriate concentrations. The cells were e x p o s e d to various c o n c e n t r a t i o n s of bleomycin (0.1 to 10/zg/ml) during their stationary phase for 8 days. The medium was changed e v e r y 2 days. When cells reached their stationary phase, the medium was changed to a fresh medium containing 5% fetal bovine serum and c y c l o h e x i m i d e ( I /zg/ml) or actinomycin D (I /zg/ml). After an 8-day treatment with bleomycin, the cells were labeled with 0 . 5 / t C i / m l of D[6-3H]glucosamine hydrochloride (sp. act., 29 Ci/m-mole) for 6 hr. Extraction of AGAG and glycoproteins. After cultivation, the medium and harvested cells were treated separately with Pronase E (Kaken Chemicals Co., Tokyo). The digest was added, with trichloroacetic acid, to Pronase E, and the precipitated protein was r e m o v e d by centrifugation. The supernatant fraction was dialyzed and the dialysate was designated as a h e x o s a m i n e - c o n t a i n i n g substance. A G A G was precipitated by adding cetylpyridinium chloride to the dialysate. The supernatant fraction was designated as the glycoprotein fraction. AGAG samples were subjected to electrophoresis on cellulose acetate strips (Separax, J o k o S a n g y o Co., T o k y o ) with 0.3 M calcium acetate. The location of H A and sulfated G A G was d e t e c t e d by staining the strip with alcian blue. Both e l e c t r o p h o r o g r a m s were cut out and counted in a scintillation counter.

1552 M o r e details 19, 101.

K. OTSUKA. S. MUROTA and Y. MORt are d e s c r i b e d

in p r e v i o u s p a p e r s

Assay of HA synthetase. A f t e r c u l t i v a t i o n , the cells were h a r v e s t e d with a teflon s p a t u l a a n d t h e n mixed v i g o r o u s l y in a T h e r m o - M i x e r ( T h e r m o n i c s Co., T o k y o ) . T h e cell l y s a t e s o b t a i n e d w e r e f r o z e n a n d t h e n t h a w e d t w o t i m e s a n d c e n t r i f u g e d at 10,000g f o r 15 rain. T h e pellet w a s w a s h e d t h r e e t i m e s with cold saline a n d s t o r e d at - 2 0 ° . T h e s a m p l e was t h a w e d by t h e a d d i t i o n of a m e a s u r e d a m o u n t of cold saline just b e f o r e e n z y m e a s s a y . A l i q u o t s w e r e t a k e n a n d the p r o t e i n c o n t e n t s meas u r e d by the m e t h o d of L o w r y et al. 1121. T h e rest of the s a m p l e w a s used as a n e n z y m e source. H A s y n t h e t a s e a c t i v i t y was a s s a y e d a c c o r d i n g to t h e m e t h o d of l s h i m o t o et al. [ 13]. T h i s e n z y m e r e a c t i o n was b a s e d on the i n c o r p o r a t i o n of U D P [ ' 4 C I N a c e t y l g l u c o s a m i n e (sp. act., 300 m C i / m - m o l e ) into H A in the p r e s e n c e of T r i s - H C I buffer (pH 7,2), U D P - g l u c u r o n i c acid, MgCI~, a n d the e n z y m e ( a b o u t 50/zg protein). T h e r e a c t i o n m i x t u r e w a s i n c u b a t e d at 37 ° f o r 60 rain a n d the r e a c t i o n s t o p p e d by p u t t i n g the r e a c t i o n t u b e s into boiling w a t e r f o r 2 min. T h e r e a c t i o n m i x t u r e w a s s p o t t e d o n W h a t m a n No, 3 M M filter p a p e r a n d s u b j e c t e d to des c e n d i n g p a p e r c h r o m a t o g r a p h y with the f o l l o w i n g s y s t e m : i s o b u t y l i c a c i d - I M N H 4 O H (5:3, v/v). A f t e r 48 hr of d e v e l o p m e n t , the r a d i o a c t i v i t y in the i m m o b i l e material r e m a i n i n g at the origin was m e a s u r e d with 10 ml of scintillation fluid (4 g PPO, 0.3 g P O P O P a n d 1 1. toluene)* by a liquid scintillation c o u n t e r . T h e r a d i o a c t i v i t y at t h e origin w a s r e d u c e d to the b a c k g r o u n d level w h e n the r e a c t i o n m i x t u r e w a s c h r o m a t o g r a p h e d a f t e r d i g e s t i o n with h y a l u r o n i d a s e f r o m Streptomyces hyalurolyticus. T h e specific a c t i v i t y of this e n z y m e w a s e x p r e s s e d as p m o l e s of [ z 4 C ] N - a c e t y l g l u c o s a m i n e i n c o r p o r ated into H A / h r / m g of p r o t e i n .

15

o.

10

[[

L/J 0

Bleomycin

Effect of bleomycin on HA synthetase activity in cultured ]ibroblasts. H A s y n t h e t a s e is k n o w n t o act at t h e final s t e p o f the p a t h w a y of H A s y n t h e s i s , a n d this e n z y m e p o l y m e r i z e s a n e q u a l molality of U D P g l u c u r o n i c acid a n d U D P - N - a c e t y l g l u c o s a m i n e to p r o d u c e H A . As e x p e c t e d , t h e H A s y n t h e t a s e activity in the c u l t u r e d f i b r o b l a s t s w a s e n h a n c e d by bleomycin. A marked increase 06 per cent over c o n t r o l ) in the a c t i v i t y of this e n z y m e w a s s e e n in cells t r e a t e d w i t h 1 /xg b l e o m y c i n (Fig. I), a n d this s t i m u l a t i o n w a s e n h a n c e d by l e n g t h e n i n g the t i m e of t r e a t m e n t , i.e. b y 48 p e r c e n t w i t h a 6-day t r e a t m e n t a n d b y 96 p e r c e n t with a n 8-day t r e a t m e n t

* PPO, 2,5-diphenyloxazole; POPOP, methyl-5-phenyloxazolyl)]benzene.

1,4-bis[2-14-

1

I

~0 #g/mr

concentrotion,

(Fig. 2). It s e e m s n e c e s s a r y f o r the cells to c o n t a c t 1 ttg b l e o m y c i n / m l f o r m o r e t h a n 4 d a y s in o r d e r to e l e v a t e significantly the e n z y m e activity, since h i g h e r d o s e s of b l e o m y c i n failed to s h o r t e n the lag time (not s h o w n ) . R e c e n t l y , it w a s r e p o r t e d that H A s y n t h e t a s e a c t i v i t y was i n d u c e d w h e n calf s e r u m was a d d e d to t h e m e d i u m [14]. As s h o w n in Fig. 3, H A s y n t h e t a s e a c t i v i t y w a s i n d u c e d w h e n the old m e d i u m of the s t a t i o n a r y c u l t u r e was r e n e w e d with f r e s h m e d i u m c o n t a i n i n g 5% fetal b o v i n e s e r u m . T h e H A syn-

/

10

J;:

O. >., .+-_ >

RESULTS AND DISCUSSION

k

Fig. 1. Dose-response curve of cultured fibroblasts treated with bleomycin. Enzyme activity was assayed after an 8-day treatment with bleomycin. Medium transfer was performed every 2 days. Cultures were harvested 6 hr after the last renewal of media, and hyaluronic acid synthetase in the cells was assayed. Details are described in the experimental section. Each point indicates mean _+S.E. (N = 3). The results indicate the pmoles/hr/mg of protein.

Reagents. C y c l o h e x i m i d e w a s p u r c h a s e d f r o m W a k o Pure C h e m i c a l I n d u s t r i e s L t d . , T o k y o , Actinomycin D was purchased from Makor Chemicals L t d . , J e r u s a l e m . U D P - g l u c u r o n i c acid w a s p u r c h a s e d f r o m B o e r i n g e r M a n n h e i m , T o k y o . All the r a d i o a c t i v e m a t e r i a l s w e r e p u r c h a s e d f r o m t h e Radiochemical Center, Amsterdam.

l Or

. .___4-

.....

>, N C L/J

I 0

'2 Time

of e x p o s u r e

I

I

I

4.

6

8

t"o b l e o m y c i n ,

days

Fig. 2. Effect of bleomycin on hyaluronic acid synthetase activity in cultured fibroblasts. Cells were exposed to I ,ug bleomycin/ml for 0-8 days during the stationary phase. Medium transfer was performed every 2 days. Cultures were harvested 6 hr after the last renewal of media, and the enzyme activity was assayed. Each point indicates mean +- S.E. (N = 3). The results indicate the pmoles/hr/mg of protein: broken line = control cultures: solid line = bleomycin-treated cultures.

1553

Bleomycin and hyaluronic acid synthetase

Table I. Direct effect of bleomycin on hyaluronic acid synthetase activity in a cell-free system*

15

Dose (,ug/ml) 0 I l0 100 Q.

Enzyme activity (pmoles/hr/mg protein) 10.75 10.03 11.07 11.24

_+0.07+ ± 1.49 + 0.07 _+0.67

* Cultures were harvested 6 hr after the last renewal of medium and the enzyme activity was measured. Enzyme fraction was obtained from control cultures. Bleomycin was added directly to the enzyme reaction mixture. + Results are expressed as mean ± S.E. (N = 3).

J=

E

Bleomycin e ~ e

0

Control

I

I

I

I

2

5

I 4

I

I

,5

6

hr

Fig. 3. Effect of medium change on hyaluronic acid synthetase activity in cultured fibroblasts treated with I u g bleomycin/ml. Enzyme activity was assayed after an 8-day treatment with bleomycin. Medium transfer was performed every 2 days. Cultures were harvested 0--6 hr after the last renewal of media, and hyaluronic acid synthetase in the cells was assayed. Each point indicates mean ± S.E. (N = 3). The results indicate the pmoles/hr/mg of protein. t h e t a s e a c t i v i t y w h i c h w a s i n d u c e d r e a c h e d a maxim u m level f o r 4 h r a n d t h e n d e c r e a s e d gradually. O n t h e o t h e r h a n d , H A s y n t h e t a s e a c t i v i t y in the g r o u p p r e t r e a t e d with b l e o m y c i n w a s significantly e n h a n c e d a n d c o n t i n u e d to i n c r e a s e d u r i n g the p e r i o d of o b s e r v a t i o n , c o m p a r e d with t h a t of the u n t r e a t e d g r o u p . T h e r e f o r e , w e s u g g e s t that the ability of the f i b r o b l a s t s f o r H A s y n t h e s i s w a s acc e l e r a t e d by t r e a t m e n t with b l e o m y c i n . H o w e v e r , H A s y n t h e t a s e a c t i v i t y in t h e old m e d i u m b e f o r e r e n e w a l did not i n c r e a s e , e v e n w h e n t h e cells w e r e t r e a t e d with b l e o m y c i n f o r 8 d a y s .

Direct effect o f bleomycin on the H A synthetase activity in cultured fibroblasts. T e t s u k a et al. [15] h a v e r e p o r t e d t h a t b l e o m y c i n e n h a n c e d the a c t i v i t y of p r o t o c o l l a g e n p r o l i n e h y d r o x y l a s e in collagen b i o s y n t h e s i s , a n d t h a t this e n h a n c e d a c t i v i t y was d u e to a n altosteric t r a n s i t i o n of the e n z y m e . In o r d e r to e x a m i n e w h e t h e r b l e o m y c i n h a s a d i r e c t effect on H A s y n t h e t a s e itself, t h e effect of bleom y c i n was e x a m i n e d in a cell-free s y s t e m . A s s h o w n in T a b l e 1, b l e o m y c i n a d d e d d i r e c t l y to t h e e n z y m e r e a c t i o n m i x t u r e s h o w e d n o s t i m u l a t o r y effect, e v e n at t h e high d o s e of 100/~g b l e o m y c i n / m l . T h i s result s u g g e s t s t h a t b l e o m y c i n d o e s not act as a n allosteric a c t i v a t o r of H A s y n t h e t a s e .

Effect of cycloheximide or actinomycin D on the stimulation o f H A synthetase activity by bleomycin.

In o r d e r to k n o w w h e t h e r s y n t h e s i s o f b o t h protein a n d R N A is i n v o l v e d in the s t i m u l a t o r y effect of b l e o m y c i n o n H A s y n t h e t a s e , i n h i b i t o r s of protein a n d R N A s y n t h e s e s were used. As s h o w n in T a b l e 2, the a c t i v i t y of this e n z y m e , in the cells t r e a t e d with c y c l o h e x i m i d e , d e c r e a s e d to a b o u t 40 per c e n t in b o t h c o n t r o l a n d b l e o m y c i n g r o u p s , a n d in the cells t r e a t e d with a c t i n o m y c i n D, the e l e v a t i o n of H A s y n t h e t a s e a c t i v i t y was also b l o c k e d c o m pletely. T h e s e r e s u l t s suggest t h a t n e w s y n t h e s i s of b o t h p r o t e i n a n d R N A is r e q u i r e d for an i n c r e a s e in H A s y n t h e t a s e activity. It h a s b e e n g e n e r a l l y r e c o g n i z e d t h a t the s y n t h e s i s o f A G A G is i n h i b i t e d by inhibitors of protein synt h e s i s since c o n c o m i t a n t s y n t h e s i s of the c o r r e s p o n d i n g c o r e p r o t e i n in the b i o s y n t h e s i s of A G A G is n e c e s s a r y [16-20]. H o w e v e r , d e t a i l e d d a t a supp o r t i n g the e x i s t e n c e of H A in a n i m a l tissue as a p r o t e o g l y c a n are lacking. In o r d e r to d e t e r m i n e w h e t h e r the inhibition of H A s y n t h e t a s e i n d u c e d in the c u l t u r e s t r e a t e d with c y c l o h e x i m i d e or a c t i n o m y c i n D was d u e to d i r e c t effects of t h e s e r e a g e n t s o n the s y n t h e s i s of this e n z y m e itself or to i n d i r e c t effects c a u s e d by a d i m i n i s h e d s y n t h e s i s of c o r e p r o t e i n , the c u l t u r e d cells w e r e labeled with [3H]glucosamine for 6 hr in t h e p r e s e n c e of c y c l o h e x i m i d e a n d t h e i r effects on t h e s y n t h e s i s of v a r i o u s h e x o s a m i n e - c o n t a i n i n g subs t a n c e s w e r e c o m p a r e d . A s s h o w n in T a b l e 3, i n h i b i t i o n of the i n c o r p o r a t i o n of [:~H]glucosamine into H A by c y c l o h e x i m i d e w a s e x c e e d i n g l y small in the c u l t u r e d f i b r o b l a s t s c o m p a r e d with the inhibition of i n c o r p o r a t i o n into c h o n d r o i t i n sulfate a n d g l y c o p r o t e i n , w h i c h h a v e b e e n r e c o g n i z e d to h a v e c o r e p r o t e i n . T h i s effect of c y c l o h e x i m i d e was i n c o n s i s t e n t with r e s u l t s o b t a i n e d b y M a t a l o n a n d D o r f m a n [21] o n c u l t u r e d fibroblast a n d by S m i t h and Hammerman[22] on mouse fibroblasts and s y n o v i a l cells. H o w e v e r , t h e i n h i b i t o r y rate f o r the s y n t h e s i s of H A s y n t h e t a s e w a s larger t h a n t h a t for the i n c o r p o r a t i o n of [ a H ] g l u c o s a m i n e into H A . F r o m t h e s e r e s u l t s it s e e m s m o s t r e a s o n a b l e to c o n c l u d e t h a t inhibition of H A s y n t h e t a s e i n d u c e d b y c y c l o h e x i m i d e is r e l a t e d to the d i m i n i s h e d synt h e s i s of H A s y n t h e t a s e r a t h e r t h a n to t h e inhibition of core protein synthesis. T h e o b s e r v a t i o n t h a t n e w m e d i u m is n e c e s s a r y for s t i m u l a t i o n of H A s y n t h e t a s e , as d e s c r i b e d a b o v e , s u g g e s t s t h a t i n d e p e n d e n t s t i m u l a t i o n of H A synt h e t a s e by b l e o m y c i n is unlikely. A c c o r d i n g l y , it c a n

K. OTSUKA, S. MUROqA and Y. MOR1

1554

"Fable 2. Effect of cycloheximide or actinomycin D on the enhancement of hyaluronic acid synthetase activity by bleomycin* Enzyme activity (pmoles/hr/mg protein) Treatment

Control

Cycloheximide

Absent Present Absent Present

Actinomycin D

6.36 2.66 7.01 3.19

+ (I.62i + 0.26 +(I.73 :* 0.07

('4

Bleomvcin

';

lt)O 42 10(I 46

12.31 + t). 15 4.43 ~ (1.43 13.12+0.52 2.8t + 0.20

II}0 36 100 22

Cells were exposed to 1 /,g/ml of cycloheximide or actinomycin D for 6 hr after an 8-da~, treatment with I ,ug/ml of bleomycin. + Results are expressed as mean ~- S.E. (N = 31.

"Fable 3. Effect of cycloheximide on the synthesis of hexosamine-containing substances by cultured fibroblasts + Glycosaminoglycan (cpm/I ,: I(1';cells l

Group Cell layer Control Cycloheximide (per cent inhibition) Medium Control Cycloheximide (per cent inhibition)

Glycoprotein [cpm/I × 10" cells)

Total

Hyaluronic acid

Sulfated glycosaminoglycan

3,036 ± 118+ 1,199 ± 13 (6(I)

17,259 ± 21 12,082 ~ 146 (3111

8.997 + 86 7,1187 + 428 (211

1.297 :~ 76 277 t 46 (791

17,934 = 1,3411 4,373 ± 335 (76)

11,430 + 132 6,708 + 96 (41 )

6,487 - 663 6,407 ± 184 t01

2.278 + 87 ~66 : 23 (84)

* Radioactivity of the extracted glycoprotein and total AGAG was measured in a liquid scintillation counter. The scintillator mixture was c o m p o s e d of toluene (I I.), Triton X-100 (500 ml) and 2.5-diphenyloxazole (6 g). The AGAG obtained was subjected to electrophoresis on cellulose acetate strips, and the radioactivity of HA and sulfated GAG on the eleclrophorogram developed with authentic standard samples was measured by a liquid scintillation counter in the conventional t o l u e n e - P P O - P O P O P system by cutting out the individual channels stained with alcian blue. + Each value indicates mean ± S.E. (N - 3). be c o n c l u d e d t h a t b l e o m y c i n a d d i t i o n a l l y e n h a n c e s H A s y n t h e t a s e activity induced by the addition of f e t a l b o v i n e s e r u m . F u r t h e r e x p e r i m e n t a t i o n is necessary to explain fully this interesting observation.

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